Silencing of the protein-coding gene IL2RG with RDE. (A) Genomic IL2RG region on chromosome Xq13.1 targeted by the ZFNs in K562 cells, a female chronic myelogenous leukemia cell line containing an active (Xa) and an inactivated (Xi) X-chromosome. The ZFNs cut in exon 5. The binding site for each zinc finger protein (ZFP) is depicted in red. The site-specific integration via HR is mediated through the left and right homology arms surrounding the integration cassette containing GFP and the RDE (here: bGH or SV40+bGH poly(A) signals). (B) Overview about possible experimental outcomes. Even heterozygous integrations of the RDE can lead to a knockout phenotype if the active X chromosome harbors the integration site. (C) IL2RG expression in individual clones after ZFN-mediated RDE integration. The IL2RG mRNA expression levels were determined by qRT–PCR for 34 wild-type clones of untreated K562 cells as well as for 216 clones with a heterozygous or homozygous RDE integration mediated by a ZFN. Individual clones displayed expression levels around or below 0.1% expression compared with the average IL2RG expression level in untreated K562 clones. (D) Average IL2RG expression after ZFN-mediated RDE integration. On average, the IL2RG mRNA expression was significantly decreased in heterozygous as well as homozygous clones compared with the expression level in wild-type K562 cells. (***) P < 0.001, t-tests.
